Abstract:

Age-related decline in the capacity of endogenous antioxidant systems
complicates pharmaceutical use by the elderly by potentially increasing the toxicity of
some compounds. Of particular interest is chemical detoxification via glutathione (GSH)
and attendant GSH-requiring detoxification enzymes, e.g. GSH peroxidases (GPX) and
GSH S-transferase (GST). These enzymes have been shown to decline in concentration
with advancing age. Thus, agents that increase peroxide formation or those that are
directly detoxified via GSH may cause enhanced toxicity in the elderly because of loss of
GSH and enzyme function.
2-methyl-1,4-naphthoquinone (menadione) is a medicinally useful precursor for
in vivo vitamin K synthesis, and this vitamin declines with age. However, menadione is
also a redox cycling agent which exerts toxicity via generation of hydrogen peroxide and
attendant oxidative damage to cellular lipids, proteins, and DNA. Thus, the clinical
benefits of providing menadione to elderly subjects may be outweighed by increased
oxidative stress and acute toxicity. In contrast, prior research has demonstrated a reversal
of peroxide-induced hepatotoxicity by dietary supplementation with (R)-a-lipoic acid
(LA), suggesting that dietary supplementation of this dithiol compound may be necessary
to ameliorate any potential age-related increase in menadione toxicity.
The two basic questions addressed in this study were: 1) Does an age-related
increase in menadione hepatotoxicity exist? 2) Does supplementation with LA protect
the cells?
Male, F344 rats in two age groups, 2-5 months and 23-24 months were used to
model young adult and elderly (-70 years) human age groups. The rats were either fed a
control diet of AIN-93M or the control diet plus LA [0.05% (w/w) or 0.1% (w/w)] for two
weeks prior to hepatocyte isolation. Following isolation, the dispersed hepatocytes were
incubated with increasing concentrations of menadione and changes in their viability was
determined by lactate dehydrogenase release. Since menadione is lipophillic, changes in
vitamin E (both a- and y-tocopherol) status were assessed to determine if these important
lipid phase antioxidants play a role in menadione detoxification.
Comparing hepatocytes from young and old unsupplemented rats revealed an agerelated
decrease in viability of up to 20%, in the populations exposed to 75 and 100 μM
menadione concentrations and strikingly similar patterns of loss in both age groups.
Similarly, both age populations displayed a biphasic response to menadione, with a
marked increase in toxicity occurring at 75 to 90 minutes in all levels except in the young
rat cells exposed to 50 μM menadione. These cells showed a monophasic rate of
viability loss during the entire two-hour time course. In the other groups, prior to the
biphasic event, or generally all time points before 75 to 90 minutes, the concentrationdependant
(50 tM to 100 [M) slope describing the loss of viability of young rat
hepatocytes changed by a factor of only 1.8, while the rate for old rat hepatocytes
changed by a factor of 2.8. In the accelerated phase, the rate of loss in young rat cells
changed by an average of4.5-fold, while the loss in old rat cells changed by 3-fold, on
average. Thus, at the 75 and 100 μM concentrations, the overall viability is lower and the
initial rate of loss is steeper for cells from old rats than that of young. Conversely, the
second phase rate of loss is steeper in cells from young rats; however, at two hours the
endpoints for both age populations at 75 and 100 μM are within 1% of each other. Thus,
although cells from young rats are initially more resistant to the toxic effects of
menadione they also suffer a precipitous decline in viability once a toxic threshold has
been reached at -75 minutes.
-25% higher baseline viability was observed in the one old LA- supplemented
(0.05% LA) rat for all time points except the 75-120 minute period at 100 ttM. An
unexpected, early decrease in viability was observed in the young LA-supplemented rats
compared to young unsupplemented animals. Vitamin E levels remained relatively
unchanged in young and old rats. Furthermore, LA supplementation did not have any
effect on vitamin E status.
Thus, within the confines of this study, we conclude that an age-related change in
susceptibility to menadione does exist in F344 rats, and that supplementation with (R)-alipoic
acid is beneficial to the old animals but possibly deleterious to the young animals.
Additionally, there was no menadione-induced reduction in the levels of hepatocellular
vitamin F.